Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14311—Parvovirus, e.g. minute virus of mice
  • C12N2750/14322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• ABSTRACT The attenuation of virulent strains of feline panleukopenia (FPL) viruses on ferret tissue and production of feline distemper (panleukopenia) vaccine 0n ferret tissue.
• panleukopenia vaccine produced on feline tissue may also carry a leukemia virus which cannot be separated from the panleukopenia vaccine.
• the objective of this invention is to provide a method of reducing the possibility of contamination of panleukopenia viruses during the attenuation stage and subsequent vaccine production stage.
• panleukopenia virus on ferret (Mustela) tissue and thereafter produce the vaccine on ferret tissue.
• the replacement of part or all of the feline tissue has the advantage that it is possible to produce attenuated virus and vaccine without contamination from other viruses normally present in feline tissue.
• the ability to employ ferret tissue is surprising from the point of view that several workers in the field have indicated that it is not possible to culture panleukopenia virus on ferret tissue.
• applicants process of attenuating panleukopenia virus comprises serially l) acclimating the virus to in vitro growth on feline tissue followed by (2) further attenuation on Mustela tissue. It is essential that acclimated virus be brought into contact with a suspension of the ferret tissue cells. Failure to bring the virus into contact with suspended Mustela cells results in no virus reproduction. The resultant attenuated virus can then be used to seed Mustela tissue production batches. In the preferred method of operation, the titer of the Mustela attenuated seed virus is increased by inocculating feline tissue with the Mustela seed virus and harvesting to obtain a Mustela/feline seed virus for Mustela tissue production batches.
• feline tissue cultures infected with a virulent strain of F PL virus are incubated for 2 to days at to C. depending upon the optimum growth rate of the cells.
• the virus laden fluids are harvested and used for subsequent passages Med. panleukopenia virus-free feline tissue cultures and ferret tissue cultures.
• the fresh tissue is obtained from disease-free animals.
• an [CK-33 feline distemper virus strain obtained from D. L. Croghan at the 33rd passage level through cats is used as the source of the virulent strain, it is desirable to serially pass the virus through about 6 to 12 passages of feline tissue.
• acclimation to in vitro growth may be attained by passing the virus through six feline lung tissue culture passages followed by two passages through feline embryo cells and two passages of feline kidney tissue.
• the last two feline kidney tissue passages are desirably employed in order to increase the virus titer.
• the virus produces a moderate fever and leukopenia indicative of feline distemper in susceptible kittens.
• the virus is sufficiently acclimated that it can be passed through ferret embryo tissue cultures and attenuated further.
• three passages on ferret tissue are sufficient to complete the attenuation and produce a seed virus.
• it is usually preferred to increase the virus titer at this stage by passing the virus through a feline kidney tissue culture passage. At this stage, the virus is attenuated, antigenic and immunogenic.
• This master seed virus can be employed to produce attenuated, antigenic and immunogenic feline panleukopenia vaccine using ferret tissue.
• the growth medium in which the cells can be cultured include Parkers 199, Eagles, Earles, etc., either with or without the additional use of antibiotics such as penicillin, streptomycin, etc.
• the cells and virus are typically incubated at a temperature of about 30 to 45C., preferably 37C. i2C.
• the culture medium can be replaced at the end of about 24 to 240 hours from the time that the cells and virus are combined.
• the pH is maintained in the range of about 6.8 to 7.6, preferably about 7.2.
• the liquid containing virus is harvested from the culture of the first stage, it is further passaged in culture media containing tissue cells (cat and ferret during the virus attenuation stage and ferret tissues in the production stage).
• the virus is contained in the growth medium of the same composition as previously specified, except for the feline tissue being replaced with Mustella tissues for a period of about 24 to 240 hours at a temperature of 30 to 45C., preferably 37C. fl".
• Mustela or ferret tissue employed in this process can be from the embryo, lung or kidneys. Generally, ferret embryo tissue is employed during both the attenuation and production cycle.
• this invention is primarily directed to the attenuation of panleukopenia virus on Mustela tissue and propagation of panleukopenia vaccine on Mustela tissue
• this invention can be employed to produce one or more batches of panleukopenia vaccine on Mustela tissue where the panleukopenia seed virus has been attenuated by any of the prior art methods, such as those described in U.S. Pat. Nos. 3,520,972; 3,293,130; etc., wherein feline tissue is used for attenuation.
• the disclosures of U.S. Pat. No. 3,520,972 of Smith, et al., and No. 3,293,130 of Slater, et al. are hereby incorporated by reference.
• it is essential that the Mustela cells must be in a suspended state (not in a monolayer) with the panleukopenia virus before there is cell growth. Failure to operate in this manner results in substantially no virus growth.
• EXAMPLE 1 One ml. of ICK-33 feline distemper virus strain obtained from Dr. D. L. Croghaii at the 33fd passage level through cats was added to a tissue culture flask having 120 square centimeters of surface area together with approximately 2 X 10 feline lung cells and 25 ml. Basal Medium Eagles containing twice the normal amounts of vitamins and amino acids, 10% fetal bovine serum, 100 units penicillin and 100 mg. of streptomycin per ml. After 5 days at 37C., the virus fluids were harvested. The preceding is deemed to be passage 1. Passages 2-6 were carried out under identical conditions except that the virus fluid harvested from the preceding step was used in the succeeding step and the growth period was varied as set forth below in Table 1.
• the virus fluids from passage 6 were purified by heating at 70C. for 30 minutes to inactivate viruses other than the feline panleukopenia virus.
• the 7th and 8th serial passages were carried out in the same manner as passage 1 except that 2 X feline embryo cells were used in place of the feline lung cells and the 7th passage was maintained at 37C. for 5 days and the 8th passage was maintained at 37C. for 5 days.
• the virus was purified by filtering through a 50 millimicron membrane filter and heated for 3% hours at 75C.
• the virus was clarified by centrifugation, filtered through a 0.45 micron membrane filter and concentrated with Carbowax m. The concentrated virus was then ultracentrifuged using a cesium chloride gradient. The l 15th 1% ml. gradient out of 130 gradients was selected for further serial passage because of its relatively high potency and purity as compared to the other gradients.
• feline kidney cells were suspended in Minimum Essential Medium Eagles having non-essential amino acids and glutamine, 10% fetal bovine serum, 100 units penicillin and 100 mg. streptomycin per ml. together with the purified 1 15th gradient virus using a 1:1 ratio of virus particles (from the preceding passage) to cells. These were maintained at 37C. for 4-7 days prior to harvesting. The virus harvested from passage 10 was tested in kittens and found to produce mild panleukopenia.
• serial passages 1 1, l2 and 13 used a virus to cell ratio of 1:1. About 2 to 5 ml. of virus from the preceding passage and 30,000,000 cells of ferret embryo tissue in Minimum Essential Medium (Eagles), with nonessential amino acids and glutamine, 10% fetal bovine serum, 100 units of penicillin and 100 mg. of streptomycin/ml were suspended in roller culture bottles,
• the titer of the virus was then increased at passage No. 14 by using feline kidney cells and virus from passage 13. After maintaining the roller bottle culture at 37C. for 8 days, the culture was freeze-thawed in the manner described in the preceding paragraph.
• the resultant fluid virus was suitable for use as a master seed stock.
• EXAMPLE 2 This example illustrates the production of a panleukopenia vaccine using ferret tissue. 50,000,000 cells of ferret embryo tissue and 50,000,000 FAlD of virus, suspended in 2 liters of Minimum Essential Medium (Eagles), with non-essential amino acids and glutamine, l0% fetal bovine serum, units of penicillin and 100 mg. of streptomicinlml, were placed in a 9 liter roller bottle.” After maintaining the culture at 37C. for 8 days, the vaccine was harvested using the freeze-thaw technique described in Example 1 yielding a vaccine capable of immunizing felines.
• Minimum Essential Medium Eagles
• the vaccine was harvested using the freeze-thaw technique described in Example 1 yielding a vaccine capable of immunizing felines.
• panleukopenia seed virus was passed through a single passage through feline kidney tissue after the virus was attenuated through a cell suspension of ferret tissue.
• panleukopenia seed virus the improvement which comprises providing a suspension of ferret cells and a virulent strain of panleukopenia virus and culturing on said suspended ferret cells said panleukopenia virus.
• panleukopenia seed virus is attenuated on feline tissue to acclimate same and then serially passaged through a cell suspension of ferret tissue.

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Cited By (10)

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Citations (3)

• 1973
  • 1973-06-04 US US367032A patent/US3892627A/en not_active Expired - Lifetime
• 1974
  • 1974-05-21 GB GB2268274A patent/GB1467503A/en not_active Expired
  • 1974-05-29 NL NL7407234A patent/NL7407234A/xx not_active Application Discontinuation
  • 1974-05-30 DE DE19742425997 patent/DE2425997A1/de not_active Withdrawn
  • 1974-06-04 BE BE145035A patent/BE815871A/xx unknown
  • 1974-06-04 FR FR7419244A patent/FR2231368B1/fr not_active Expired

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